The present invention relates generally to methods and devices for carrying out specific binding assays. More specifically, the invention relates to methods for the use of chromatographic transport for the movement of reagents and reactive sample components.
The use of specific binding assays has been found to be of great value in a variety of clinical and other applications. Such assays involve the detection and determination of an analyte substance which is a member of a specific binding pair consisting of a ligand and a receptor. The ligand and the receptor are related in that the receptor specifically binds to the ligand, being capable of distinguishing the ligand from other sample constituents having similar characteristics. Specific binding assays include immunological assays involving reactions between antibodies and antigens, DNA and RNA hybridization reactions and other specific binding reactions such as those involving hormone and other biological receptors. Specific binding assays may be practiced according to a variety of formats well known to the art. Such assays include competitive binding assays, "direct" and "indirect" sandwich assays and agglutination assays.
Because the results of specific binding reactions are generally not directly observable, various techniques have been devised for labelling one member of the specific binding pair in order that the binding reaction may be indirectly observed. Useful labels include radiolabels, chromophores and fluorophores the presence of which may be detected by means of radiation detectors, spectrophotometers or the naked eye. Where members of a specific binding pair are tagged with an enzyme label, their presence may be detected by the enzymatic activation of a reaction system including a signal generating substrate/cofactor group wherein a compound such as a dyestuff, is activated to produce a detectable signal.
Specific binding assay devices are known in the art comprising vertically arranged elements including (a) a porous capture material which is impregnated at a reaction site with member of a specific binding pair such as an antibody or an antigen; (b) a removable prefilter disposed above the capture material and (c) a blotter disposed below the capture material. A sample liquid such as blood, serum or other biological fluid is added to the device wherein the prefilter removes particulates and other impurities from the sample which would otherwise be trapped on top of the specific binding capture material. Analyte substances within the sample are trapped by means of specific binding reactions with their specific binding partners on the capture material. Non-analyte components of the sample solution pass through the capture material and are absorbed by the blotter. Wash steps may be carried out to remove non-analyte components from the capture material and additional reagents such as enzyme substrates, cofactors and dye precursors, may be added to the capture material in order to indicate the presence or absence of analyte at the reaction site. The prefilter must then be removed in order that the presence or absence of analyte at the reaction site may be visually determined. Such assay devices are rapid and generally reliable but suffer from limitations in capture efficiency and sensitivity because most of the analyte in the sample material flows around rather than through the reaction site on the capture material.
Various disclosures are of interest to the present application. Tom, et al., U.S. Pat. No. 4,366,241 discloses an immunoassay device comprising a relatively small test zone including a specific binding reagent, and a relatively large absorbing zone in liquid receiving relationship with said immunosorbing zone. Immunoassays for determining the presence of an analyte material are carried out by contacting the assay device with a sample solution, a solution containing enzyme labelled specific binding material and a solution containing an enzyme catalyzed signal system. The solutions migrate through the immunosorbing zone into the liquid absorbing zone and the presence of analyte in the sample material is indicated by enzyme activation of the signal system.
Deutsch, et al., U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537 relate to immunological and other types of specific binding assays wherein reagents are transported by chromatographic solvent transport. According to one embodiment, a radiolabelled competitive binding assay kit comprises a strip capable of transporting a developing liquid by capillarity having a first zone for receiving a sample, a second zone impregnated with a first reagent capable of being transported by the developing liquid and a third zone impregnated with a second reagent. In addition, the devices comprise a measuring zone and a retarding element which may be either the second reagent or the material of the strip. The first reagent is capable of reacting with one of the group consisting of (1) the sample, (2) the sample and the second reagent, and (3) the second reagent in competition with the sample, to form a product in an amount dependent on the characteristic being determined. A sample is contacted with the first zone and the strip is then dipped into the developing liquid to bring about transport of the sample and the first reagent to form the reaction product. The retarding element slows transport of either the product or the first reagent (the moving reagent) to spacially separate the two and the amount of the moving element is then measured at the measurement location.
Also of interest to the present invention is the disclosure of co-owned and copending U.S. patent application Ser. No. 912,878 filed Sept. 29, 1986 by Gordon, et al., which is hereby incorporated by reference and which relates to devices for conducting specific binding assays utilizing the sequential chromatographic transport of analyte and reagent materials. Wash and addition steps are inherently carried out and liquid "microcircuitry" can be programmed to carry out a variety of multistep procedures and to avoid the premature mixing of sample materials and reagents. Specifically, the Gordon, et al., sequential transport application relates to devices which comprise a test strip for the detection of an analyte in a sample comprising a length of chromatographic material having the capacity for rapid chromatographic solvent transport of non-immobilized reagents and reactive sample components by means of a selected chromatographic solvent.
Of further interest to the present invention are the disclosures of Piasio, et al., U.S. Pat. No. 4,255,575 and Litman, et al., U.S. Pat. No. 4,391,904. These references relate to specific binding assay methods generally and disclose immobilization of enzyme substrates buffers and cofactors on solid phase matrices.